A beta-galactosidase gene from Bifidobacterium breve B24 which showed the higher hydrolytic and synthetic activity was cloned in E. coli. The complete beta-galactosidase gene contained 2076 bp nucleotides and encoded 691 amino acids which had a high homology to the other Bifidobacterium species. This beta-galactosidase was homologous to that of the LacA family. The galA gene was successfully over-expressed in E. coli ER2566. To observe any change in the recombinant enzyme, beta-galactosidases from Bifidobacterium breve B24 and recombinant E. coli ER2566 were purified to homogeneity by ion exchange chromatography (Mono-Q) and gel-filtration chromatography (Superose-12 and Superdex 200) columns. The molecular mass of both beta-galactosidases was estimated to be 75 kDa on SDS-PAGE. Activity staining on non-denaturing Native-PAGE and Superose-12 gel-filtration chromatography showed that the enzymes are composed of a dimer with a molecular mass of 150 kDa. / The optimum pHs of the native and recombinant enzymes for hydrolyzing O-nitrophenyl-beta-D-galactopyranose (ONPG) were pH 6.0 and 7.0, respectively, and they were stable over the pH range of 5-8 and 6-9, respectively. The optimum temperature of both enzymes for hydrolyzing ONPG was similar at 45 °C and they were stable over the temperature range of 20-45 °C. Both enzymes were stable up to 45 °C during 5 h of incubation at pH 6.5. The recombinant enzyme was slightly activated by bivalent metal ions, Mg2+, Mn2+, and Zn2+ at 1 mM but strongly inhibited by Hg2+ and p-chloromercuribenzoic acid (PCMB). The K m values of both native and recombinant beta-galactosidases for ONPG were 2.77 and 1.82 mM, respectively, and the Vmax values were 1.02 and 1.39 mM/min, respectively. / The two beta-galactosidase activities were also tested with lactose as substrate. The optimum pH of the native and recombinant enzymes for hydrolyzing lactose was similar at pH 6.0. Both enzymes had more than 80 % of their activity in the range of pH 6-8, indicating that the enzymes were stable at neutral pH. However, the native beta-galactosidase had around 40 % of its activity at pH 5.0, whereas the recombinant enzyme had no activity at this pH. On the other hand, the recombinant enzyme had over 50 % of its activity at pH 9.0, while the native beta-galactosidase showed lower than 5 % of its activity. The optimum temperature of both enzymes was at 45 °C. The profiles of both enzyme activities were very similar except at the temperature of 10 °C. The recombinant beta-galactosidase still had around 20 % of its enzyme activity at 10 °C, while no enzyme activity from the native enzyme was detected at this temperature.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.85658 |
| Date | January 2005 |
| Creators | Yi, Sung Hun, 1971- |
| Publisher | McGill University |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Format | application/pdf |
| Coverage | Doctor of Philosophy (Department of Food Science and Agricultural Chemistry.) |
| Rights | All items in eScholarship@McGill are protected by copyright with all rights reserved unless otherwise indicated. |
| Relation | alephsysno: 002223638, proquestno: AAINR12965, Theses scanned by UMI/ProQuest. |
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